This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Despite intensive study, the cholinergic nervous system is poorly understood at the molecular level. Since defects in cholinergic function have been implicated in human disorders, such as Alzhiemer's disease and congenital myasthenic syndromes, a more complete knowledge of the molecular mechanisms that control the cholinergic nervous system might contribute to a better understanding of the mechanisms involved in these disorders. The experiments outlined in this proposal are designed to continue our analysis of the cholinergic locus (cha-1 - unc-17) in the soil nematode, Caenorhabditis elegans. The cha-1 gene encodes choline acetyltransferase (ChAT), required for acetylcholine biosynthesis, and the unc-17 gene encodes the vesicular acetylcholine transporter (VAChT), which is necessary for synaptic vesicle loading. These genes comprise a cholinergic "operon" with a novel structure that is conserved from nematodes to mammals, suggesting that the overall structure may be important for proper regulation. We have already analyzed the upstream promoter region of this locus, and have identified and characterized specific regulatory sequences required for expression in subsets of cholinergic neurons. In addition, we now have evidence for a cha-1-specific promoter in C. elegans, expression from which produces a previously unidentified isoform of ChAT (CHA-1B). We now wish to use the information and tools we have developed to analyze the regulation of these genes and the contribution of the different isoforms of the ChAT to cholinergic function. The specific aims of the research are: (1) To identify proteins required for regulation of the cha-1 - unc-17 complex. (2) Study the individual contributions of the CHA-1A (expressed from the upstream promoter) and CHA-1B isoforms to cholinergic function.